The goal of this project is to use a fluorescent protein FRET-based assay to screen for selective small molecule inhibitors of the SUMO protease SENP1. This enzyme is uniquely upregulated in advanced prostate cancer and plays a key role in promoting malignant proliferation by promoting the transcriptional activity of the androgen receptor and suppressing the cellular senescence program. SENP1 is therefore an attractive and novel therapeutic target in advanced prostate cancer, an incurable disease that is the second leading cause of death for males in America. Despite its importance, there are no reported small molecule inhibitors of SENP1. The available activity-based probes are large derivatives of SUMO proteins unsuitable for drug development. A focused HTS campaign is a logical step to address this shortcoming. We propose to use a sensitive, simple and robust FRET based SENP1 assay we have developed and perform a high throughput screen to identify novel classes of compounds that are effective inhibitors of SENP1. Cell permeable selective inhibitors of SENP1 would be powerful tools to identify and manipulate the SUMOylation levels of SENP1 substrates in intact cells. They would allow a direct assessment of the functional impact of SENP1 on specific targets such as the androgen receptor. This is important because although RNA knockdown of individual SENPs is possible, the long-term nature of such experiments makes it difficult to evaluate acute changes in SUMO modification without compensatory cellular mechanisms. This class of compounds would overcome such problems. Furthermore, the pharmacologic manipulation of SENP1 activity will allow a clear assessment of the potential of SENP1 based therapeutic approaches for prostate cancer. To achieve these goals we propose as our first aim to implement a high-throughput screen to identify and confirm hit compounds. This will involve a primary screen and a tandem confirmatory screen using an orthogonal assay. As a second aim, we will optimize and characterize initial hits in order to identify potent and selective SENP1 inhibitors. To this end, we will use an iterative process of structure/activity analysis, coupled with multiple counter-screens using assays for related SUMO proteases. In a third aim, we will examine the ability of the best candidate compounds to regulate SENP1 activity in intact cells and to characterize their effects on the viability of normal and transformed prostate cell lines. We believe that the unique function and regulation of SENP1 in prostate cancer coupled with its favorable properties for drug development make a compelling case for targeting this enzyme for the discovery and development of small molecule inhibitors. Such tools can be very valuable to understand the broad biological function of SENP1 and SUMOylation and as leads for the potential development of novel prostate cancer therapeutics.